Non-Hodgkin Lymphoma (NHL) is the seventh most common cancer in the United States, with diffuse large B-cell lymphoma (DLBCL) being the most common subtype. The standard treatment of DLBCL is a cocktail of chemotherapeutic agents called R-CHOP, but roughly 10% of DLBCL patients will be refractory to this therapy, and a substantial 40% of DLBCL patients who do respond to R-CHOP will relapse within 5 years. By gene expression profiling, patients with DLBCL can be categorized into two clinically relevant subtypes: activated B-cell (ABC) DLBCL and germinal center B-cell (GCB) DLBCL. Patients with ABC DLBCL have a much worse overall survival and progression-free survival in response to standard chemotherapy than patients with GCB DLBCL. Chronic, overactive signaling through the B-cell receptor (BCR) and the anti-apoptotic NF-?B pathway are hallmarks of the ABC but not the GCB DLBCL subtype, and could therefore be particularly important for promoting tumor progression and drug resistance. The Src family kinases (SFKs) are a family of nonreceptor tyrosine kinases that are important for signaling through the BCR. The proteasome is crucial for facilitating the NF-?B signaling pathway, as it degrades the negative regulators of NF-?B signaling. Both proteasome inhibitors and inhibitors of SFK signaling targets have shown significant promise in clinical trials for the treatment of ABC DLBCL. While there is some evidence that the SFKs may play a role in regulation of the proteasome, the direct effects of SFK modulation on the proteasome are not well-studied. This proposal aims to study the role of the SFKs in the regulation of the proteasome through the use of novel peptide probes. A set of three distinct, sensitive peptide sensors have been developed to simultaneously measure the chymotrypsin-like, trypsin-like, and caspase-like activities of the proteasome. A peptide sensor has also been developed to measure the catalytic activity of the SFKs. Preliminary studies demonstrate a dose-dependent reduction in proteasome activity in response to the SFK inhibitor dasatinib. Thus, the hypothesis of this proposal is that the SFKs activate the proteasome. The SFKs will be inhibited in DLBCL cells through the use of small molecule inhibitors and siRNA-mediated knockdown, after which changes in proteasome activity, protein expression, gene expression, and protein post-translational modifications will be assessed. Subsequent studies will examine the same changes after activation of one or more of the SFK members through expression of constitutively active constructs. This work will provide valuable insight into the SFK-proteasome signaling axis in DLBCL, and will therefore provide a better understanding of the cellular processes that contribute to tumor progression and drug resistance in DLBCL. The findings of this study could lead to the identification of additional therapeutic targets for the treatment of relapsed and refractory DLBCL.